Express protein
Set up crystal trays (use 24 well plates, start with condition listed in the PDB article and vary a little bit up and down in concentration)
ICM Virtual Screening (maybe Vina screen too? - get Christina's help)
Inhibition Assays
Determine IC50 of compound
Then compare best ICM ligand to best GOLD ligand in the wetlab
Week 14:
Since there were three proteins I collected from the FPLC, I did an enzyme assay for each one of them, however there was no enzyme activity. The OD reading was 0 for the most part
I also did an enzyme assay for Dr.B's PSTP sample prior to FPLC. Again, my enzyme was inactive.
I did an enzyme assay of all three increasing the concentration of enzyme I added to 10ng/ul. Since the solution didn't turn yellow, my enzyme was inactive.
Finally, I did an enzyme assay for my PSTP sample that was snap frozen, which didn't work as well.
Figure 1: Concentration of PSTP that was snap frozen in -80 degrees Celsius
Figure 2: Concentration of Dr. B's PSTP before FPLC
Figure 3: Concentration of protein 1 in 20% glycerol
Concentration of protein 3 in 20% Glycerol - Possibly PSTP?
Week 13:
Protein Expression
skipped characterization
FPLC
concentrated protein to 0.5ml and stored in 20% glycerol
Figure 1: Concentration of Elution 1 sample after purification
Figure 2: Concentration of Elution 2 Sample after Purification
Figure 3: FPLC graph
Week 12:
Enzyme Assay (2 Trials)
1st trial - PTP1b as a control; used 60ul and 120ul of enzyme to test activity
PTP1b enzyme served as the positive control - turned yellow upon addition of pNPP substrate so enzyme works
PSTP did not turn yellow and the absorbance readings were 0.
2nd trial - supplemented enzyme with manganese ions (final concentration is 1mM)
Tube A with 60ul of enzyme: 0.148 (absorbance)
Tube B with 120ul of enzyme: 0.107 (absorbance)
Need to do protein expression again...
Week 11: S - since the activity is below the control, the enzyme may not be active. I think the big bar in the second graph is an outlier. - May need to think about regrowing enzyme. Dr. B. (also may be worth comparing to PTP1b in same assay)
ACS
symBIOsis
Virtual Screening
Enzyme Inhibition Assay with and without TCEP
Figure 1: Enzyme inhibition assay test without TCEP
Figure 2: Enzyme Inhibition Assay with TCEP
Week 10:
Virtual Screening-working on primary and secondary runs for MW-set_3D.sdf Chembridge Library
Figure 1: Best ranking list from first run of cb_306.
Week 9:
Enzyme Test
Figure 1: Absorbance of protein measured at 410nm
Figure 2: Enzyme Activity - Absorbance at 410 nm vs Concentration of protein (nM).
Concentrated protein after FPLC (below are the concentrations after FPLC)
Figure 2: Concentration of PSTP after it was diluted in buffer during FPLC - the concentration after Beer's Law calculation was 3.2uM.
Figure 3: Concentration of unknown protein detected by FPLC
Figure 4: Concentration of PSTP after concentrating it to 16uM (After FPLC) - the concentration after Beer's Law calculation was 10.5 uM
stored half of sample (.5ml) in 20% glycerol and snap froze the other half using liquid nitrogen
Crystallography workshop
Week 8:
Virtual Screened HF9, cd_306, MW-set libraries.
concentrated protein - combined elution 1 and 2 samples
Figure 1: After concentrating protein-before washing the "walls" of the filter.
Figure 2: After concentrating protein - after washing the "walls" of the filter.
Sadhana - embed this image instead of putting it as a link..... Dr. B
Week 7: - Need images or Data - Dr. B
Virtual screening on PSTP - screened the HF9 library
worked on pymol image of PSTP
Dried characterization gel
lab report
Week 6: (YEAH, Sadhana..... - good expression - Dr. B)
protein purification - combined soluble fraction of tube A and tube B
protein characterization (ran gel)
lane 1: protein ladder
lane 2: sample 1 (post induction) flask A
lane 3: sample 1 (post induction) flask B
lane 4: sample 2 (soluble fraction) tube A
lane 5: sample 2 (soluble fraction) tube B
lane 6: sample 3 (flow through)
lane 7: sample 4 (wash)
lane 8: sample 5 (Elution 1)
lane 9: sample 6 (Elution 2)
Figure 1: Page gel (protein characterization); Sample 1 in lanes 1 and 2 look like they are the elution samples..sample 1 is supposed look like samples 2, 3, and 4 because it should technically have many other proteins.
Concentrations of Elution Samples:
Figure 2: Concentration of elution 1 sample (280nm)
Figure 3: Concentration of Elution 2 sample (280nm)
Week 5: - Sadhana - show some data - what were the pellet weights??
Made page gel
protein expression continued: lysis step 4
Week 4: (Sadhana - show a screen shot of your BLAST image instead of posting a link to a WORD doc. - Thanks,DrB.)
DNA sequencing results: Samples 2 and 6 matched completely. Sample 5 had a 99% match in both the forward and reverse sequence. I will be using Sample 6 for protein expression because it had the highest concentration. (37.8ng/ul)
Transformation (Bl21 competent cells) - How many colonies? - Dr. B (46 colonies on plate A and 221 colonies on plate B)
Protein Expression - What was the pellet weight? - Dr. B (3.08g and 2.96g)
Figure 1: The top 2 plates are test plates with competent cells that had been left outside. The bottom two plates are my transformation plates with BL21 competent cells and my protein, PSTP. Plate A in the bottome ledt has 46 colonies and plate B on the bottom right has 221 colonies.
Figure 2: My transformation plates and overnight culture.
Week 3:
Centrifuged samples and retrieved the pellet (stored in -20 degrees Celsius)
Miniprep
Sent 3 samples (2,5,6) to DNA sequencing- The other samples had very low concentrations because I eluted with 100 ul of water as opposed to 50 ul of water.
Figure 1: Lane 1-skip, Lane 2-uncut pNIC, Lane 3-cut pNIC...Bsa1 did not cut pNIC....possibly due to an error in PCR cleanup
Week 1:
Ran secondary PCR on gel
Ran PCR Squared
PCR Cleanup on PCR Squared Sample
Figure 1: Concentration of secondary PCR prior to PCR cleanup
Figure 2: Secondary PCR prior to PCR cleanup; Lane 1: 100bp DNA ladder; Lane 2: Secondary PCR
Figure 3: Concentration of PCR squared sample after PCR clean-up.
Figure 1: 1ng of Plasmid pGFP was incorporated into the DH5 bacteria. A total of 240 colonies were grown
Figure 2: 5ng of Plasmid pGFP was incorporated into the DH5 bacteria. A total of 424 colonies were grown.
Figure 3: 25ng of Plasmid pGFP was incorporated into the DH5 bacteria. A total of 784 colonies were grown
Figure 4: Restriction Enzyme Digest Gel; Lane 2 is the DNA ladder, Lane 3 is the uncut plasmid, Lane 4 is EcoR1, Lane 5 is PvuII, and Lane 6 is EcoR1 + PvuII.
Figure 5: pmCherry gel; no bands present....possibly due to an error in the PCR or pipetting.
Figure 6: pGBR22 gel; Lane 1: skip, Lane 2: DNA ladder, Lane 3,4,5,6 Samples.
Sadhana's Research
Express protein
Set up crystal trays (use 24 well plates, start with condition listed in the PDB article and vary a little bit up and down in concentration)
ICM Virtual Screening (maybe Vina screen too? - get Christina's help)
Inhibition Assays
Determine IC50 of compound
Then compare best ICM ligand to best GOLD ligand in the wetlab
Week 14:
Week 13:
Week 12:
Week 11: S - since the activity is below the control, the enzyme may not be active. I think the big bar in the second graph is an outlier. - May need to think about regrowing enzyme. Dr. B. (also may be worth comparing to PTP1b in same assay)
Week 10:
Week 9:
Week 8:
Week 7: - Need images or Data - Dr. B
Week 6: (YEAH, Sadhana..... - good expression - Dr. B)
Concentrations of Elution Samples:
Week 5: - Sadhana - show some data - what were the pellet weights??
Week 4: (Sadhana - show a screen shot of your BLAST image instead of posting a link to a WORD doc. - Thanks,DrB.)
Week 3:
Sadhana - looks good. - Dr. B
Week 2:
Week 1: